The overall objective of this proposal is to elucidate the mechanism of early and late preconditioning against lethal ischemia. Our major hypothesis is that a mild increase in intracellular Ca++ effected by experimental and pharmacological interventions is a strong trigger for preconditioning and protein kinase C is a major player in the signaling cascade leading to protection. The opening of mitochondrial KATP channel which is believed to be pivotal in protection is dependent on the activation of PKC by intracellular [Ca++]i increase. Mitochondria which occupy a third of total cellular volume and perform multifactorial functions play a significant role in the survival of myocytes in stress and ischemic conditions. Under immediate stress conditions, the opening of mitochondrial KATP channel maintains the cell integrity by regulation of Ca++ homeostasis and increased ATP synthesis while the delayed protection is mediated by augmented synthesis of antioxidants. Both cell cultures and intact hearts will be used. The specific aims are to: determine whether [Ca++]i fluctuations by oxidative stress elicit both early and late protection; whether [Ca++]i mediated activation of PKC is responsible for early and late PC; whether opening of mitochondrial KATP channel is important in protection in late PC; whether opening of mitochondrial KATP channel is dependent on PKC activation and its translocation to mitochondria during early and late PC; if early and late PC by [Ca++]i reduces cell injury by inhibiting apoptosis by regulating permeability transition; if late PC by [Ca++]i is due to synthesis of antioxidants which inhibits apoptosis. A wide range of multidisciplinary techniques, including biochemistry, cell biology, molecular biology, electron microscopy, immunocytochemistry and pharmacological approaches will be used to integrate the information at the cell level with the function at the organ level. This investigation will produce important new insights into the molecular mechanisms of preconditioning and will have important implications for designing therapeutic interventions based on the unique endogenous adaptive responses by the myocardium.